Synthesis and Evaluation of 68Ga-Labeled (2S,4S)-4-Fluoropyrrolidine-2-Carbonitrile and (4R)-Thiazolidine-4-Carbonitrile Derivatives as Novel Fibroblast Activation Protein-Targeted PET Tracers for Cancer Imaging

Fibroblast activation protein α (FAP-α) is a cell-surface protein overexpressed on cancer-associated fibroblasts that constitute a substantial component of tumor stroma and drive tumorigenesis. FAP is minimally expressed by most healthy tissues, including normal fibroblasts. This makes it a promising pan-cancer diagnostic and therapeutic target. In the present study, we synthesized two novel tracers, [68Ga]Ga-SB03045 and [68Ga]Ga-SB03058, bearing a (2S,4S)-4-fluoropyrrolidine-2-carbonitrile or a (4R)-thiazolidine-4-carbonitrile pharmacophore, respectively. [68Ga]Ga-SB03045 and [68Ga]Ga-SB03058 were evaluated for their FAP-targeting capabilities using substrate-based in vitro binding assays, and in PET/CT imaging and ex vivo biodistribution studies in an HEK293T:hFAP tumor xenograft mouse model. The IC50 values of natGa-SB03045 (1.59 ± 0.45 nM) and natGa-SB03058 (0.68 ± 0.09 nM) were found to be lower than those of the clinically validated natGa-FAPI-04 (4.11 ± 1.42 nM). Contrary to the results obtained in the FAP-binding assay, [68Ga]Ga-SB03058 demonstrated a ~1.5 fold lower tumor uptake than that of [68Ga]Ga-FAPI-04 (7.93 ± 1.33 vs. 11.90 ± 2.17 %ID/g), whereas [68Ga]Ga-SB03045 (11.8 ± 2.35 %ID/g) exhibited a tumor uptake comparable to that of [68Ga]Ga-FAPI-04. Thus, our data suggest that the (2S,4S)-4-fluoropyrrolidine-2-carbonitrile scaffold holds potential as a promising pharmacophore for the design of FAP-targeted radioligands for cancer diagnosis and therapy.


Introduction
Fibroblast activation protein α (FAP-α) is a type II transmembrane protein belonging to the family of non-classical serine proteases, and it cleaves Pro-Xaa peptide bonds [1]. FAP is a member of the "DPPIV-like" (DPP, dipeptidyl peptidase) class of enzymes, and possesses both dipeptidyl aminopeptidase and endopeptidase activities [1,2]. FAP is overexpressed on the cell surface of cancer-associated fibroblasts (CAFs), a type of continuously activated fibroblasts. CAFs constitute a dominant stromal component accounting for up to 80% of all stromal fibroblasts in the tumor microenvironment [2]. This overexpression is seen in the reactive stromal fibroblasts of most carcinomas, including prostate and breast cancers [3,4]. Studies have linked this overexpression to enhanced tumorigenic potential, tumor growth rates, and poor prognosis, with the association being more prominent if overexpression is found in the cancer cells rather than in the stroma [5,6]. In addition to CAFs, high levels of FAP have also been noted at tissue remodeling interfaces of cirrhotic liver [7], In this regard, nat Ga-complexed SB03045 and SB03058 were evaluated in vitro using a substrate-based fluorescence assay to determine their FAP-inhibitory capabilities. Their 68 Ga complexed analogs, on the other hand, were subjected to PET/CT imaging and ex vivo biodistribution studies using an HEK293T:hFAP tumor xenograft mouse model, and LogD7.4 partitioning studies. The results were then compared with those obtained using [ 68 Ga]Ga-FAPI-04, either reported previously [30] or head-to-head in the present study.

Synthesis of 68 Ga and nat Ga-Complexed DOTA-Conjugated FAP-Targeted Ligands
Detailed information on the synthesis, purification, and characterizations of precursors and nat Ga-complexed is provided in the Supplementary Materials ( Figures S1-S6).
Multistep organic syntheses of DOTA-conjugated precursors SB03045 and SB03058 are presented in Schemes 1 and 2, respectively. Briefly, Compound 1 was prepared following literature procedures [24] and reacted with 2,3,5,6-tetrafluorophenol (TFP) to obtain its activated TFP-ester 2 in 61% yield [31]. Compound 2 and Compound 3 [14], prepared following literature procedures, were coupled overnight to obtain compound 4 in 63% yield. Compound 4 was first Boc-deprotected using 50% trifluoroacetic acid (TFA) in dichloromethane (DCM), then reacted with DOTA-NHS to afford the desired precursor SB03045 in 54% yield.  In this regard, nat Ga-complexed SB03045 and SB03058 were evaluated in vitro using a substrate-based fluorescence assay to determine their FAP-inhibitory capabilities. Their 68 Ga complexed analogs, on the other hand, were subjected to PET/CT imaging and ex vivo biodistribution studies using an HEK293T:hFAP tumor xenograft mouse model, and LogD 7.4 partitioning studies. The results were then compared with those obtained using [ 68 Ga]Ga-FAPI-04, either reported previously [30] or head-to-head in the present study.

Synthesis of 68 Ga and nat Ga-Complexed DOTA-Conjugated FAP-Targeted Ligands
Detailed information on the synthesis, purification, and characterizations of precursors and nat Ga-complexed is provided in the Supplementary Materials (Figures S1-S6).
Multistep organic syntheses of DOTA-conjugated precursors SB03045 and SB03058 are presented in Schemes 1 and 2, respectively. Briefly, Compound 1 was prepared following literature procedures [24] and reacted with 2,3,5,6-tetrafluorophenol (TFP) to obtain its activated TFP-ester 2 in 61% yield [31]. Compound 2 and Compound 3 [14], prepared following literature procedures, were coupled overnight to obtain compound 4 in 63% yield. Compound 4 was first Boc-deprotected using 50% trifluoroacetic acid (TFA) in dichloromethane (DCM), then reacted with DOTA-NHS to afford the desired precursor SB03045 in 54% yield. In this regard, nat Ga-complexed SB03045 and SB03058 were evaluated in vitro using a substrate-based fluorescence assay to determine their FAP-inhibitory capabilities. Their 68 Ga complexed analogs, on the other hand, were subjected to PET/CT imaging and ex vivo biodistribution studies using an HEK293T:hFAP tumor xenograft mouse model, and LogD7.4 partitioning studies. The results were then compared with those obtained using [ 68 Ga]Ga-FAPI-04, either reported previously [30] or head-to-head in the present study.

Synthesis of 68 Ga and nat Ga-Complexed DOTA-Conjugated FAP-Targeted Ligands
Detailed information on the synthesis, purification, and characterizations of precursors and nat Ga-complexed is provided in the Supplementary Materials ( Figures S1-S6).

Ex Vivo Biodistribution and PET/CT Imaging Studies
The HEK293T:hFAP tumor xenografts [30] were clearly visualized in PET/CT images acquired at 1 h pi using both [ 68 Ga]Ga-SB03045 and [ 68 Ga]Ga-SB03058 ( Figure 3). Both tracers were excreted mainly via the renal pathway, as was apparent from the high accumulation in the urinary bladder. The tumor uptake of [ 68 Ga]Ga-SB03058 was found to be lower, whereas that of [ 68 Ga]Ga-SB03045 was found to be comparable to that of the previously reported [ 68 Ga]Ga-FAPI-04 [30].

Ex Vivo Biodistribution and PET/CT Imaging Studies
The HEK293T:hFAP tumor xenografts [30] were clearly visualized in PET/CT ima acquired at 1 h pi using both [ 68 Ga]Ga-SB03045 and [ 68 Ga]Ga-SB03058 ( Figure 3). Both t ers were excreted mainly via the renal pathway, as was apparent from the high accu lation in the urinary bladder. The tumor uptake of [ 68 Ga]Ga-SB03058 was found to lower, whereas that of [ 68 Ga]Ga-SB03045 was found to be comparable to that of the pr ously reported [ 68 Ga]Ga-FAPI-04 [30]. Although both tracers displayed fast clearance from most normal organs/tissues, background uptake of [ 68 Ga]Ga-SB03058 was slightly higher (primarily in the skel muscles and bones) than that of [ 68 Ga]Ga-SB03045, resulting in a slightly inferior mor/background contrast. Co-injection of [ 68 Ga]Ga-SB03045 with nat Ga-FAPI-04 mg/mouse) reduced the tumor uptake to almost background level ( Figure 3).
Biodistribution studies ( Figure 4 and  Table S1). In general, [ 68 Ga]Ga-SB03058 had ~1.5 fold lower tumor uptake Although both tracers displayed fast clearance from most normal organs/tissues, the background uptake of [ 68 Ga]Ga-SB03058 was slightly higher (primarily in the skeletal muscles and bones) than that of [ 68 Ga]Ga-SB03045, resulting in a slightly inferior tumor/background contrast.

Discussion
In the present study, we synthesized two novel derivatives of the clinically validated tracer  [24,26] and found in FAPI-04, was retained in both SB03045 and SB03058. Finally, the addition of chelator DOTA allowed the formation of stable complexes with the PET isotope 68 Ga. Modification was only made either to the C4 substituent in the 2-cyanopyrrolidine ring (as in SB03045) or the C4 position of 2-cyanopyrrolidine ring (as in SB03058), as rationalized below.
Structural modeling has shown that FAP contains a well-defined, hydrophobic S1 binding pocket that prefers a P1 proline with an electrophilic 2-substituent (2-cyanopyrrolidine or 2-Boro-Pro) that can form a covalent adduct with the catalytic serine nucleophile [34]. Furthermore, the S1 pocket only tolerates P2 amino acids (Gly and D-Ala) that are small enough to evade steric clashes between FAP and P3 residue or P3 residue's carbonyl group.
A series of pseudodipeptides based on a 3-aminoacyl-4-cyanothiazolidide scaffold (K i ≤ 5 nM) were reported to be very potent inhibitors of DDP-IV [29]. In particular, 3-isoleucyl-4-cyanothiazolidine (K i = 0.41 nM) was demonstrated to have >5-fold higher binding affinity than its (2S)-2-cyanopyrrolidine counterpart (K i = 2.2 nM). DPP-IV is a serine protease that hails from a class of prolyl peptidases [1,36,37]. However, unlike FAP, it is strictly a dipeptidyl exopeptidase and is only able to cleave dipeptides containing proline at the penultimate position from the N-terminus of peptides or proteins [1]. Accordingly, we substituted the pyrrolidine ring in FAPI-04 with a thiazolidine ring to yield our second derivative, SB03058, containing a (4R)-thiazolidine-4-carbonitrile binding moiety that was investigated as another novel FAP-targeted pharmacophore. We rationalized that the addition of N-(4-quinolinoyl)-glycyl fragment to the (4R)-thiazolidine-4-carbonitrile pharmacophore would impart selectivity toward FAP over its phylogenetically related dipeptidyl exopeptidase counterpart DPP-IV, which is unable to recognize N-blocked/acylated peptides.
The average IC 50 value of nat Ga-SB03045 (1.59 ± 0.45 nM) was marginally lower, whereas that of nat Ga-SB03058 (0.68 ± 0.09 nM) was found to be less by an order of magnitude than nat Ga-FAPI-04 (4.11 ± 1.42 nM) in the FAP-binding assays. Thus, the FAP-binding potency of the mono-fluorinated analog was found to be slightly better, whereas that of the thiazolidine version was observed to be >5 fold higher than the di-fluorinated analog. This confirmed our hypothesis that replacing the (2S)-4,4-difluoropyrrolidine-2-carbonitrile moiety in FAPI-04 with (2S,4S)-4-fluoropyrrolidine-2-carbonitrile or (4R)-thiazolidine-4carbonitrile could lead to new derivatives with improved FAP-binding affinity.
PET/CT images acquired at 1 h pi showed clear visualization of both [ 68 Ga]Ga-SB03045 and [ 68 Ga]Ga-SB03058, which were excreted mainly via the renal pathway ( Figure 3). The data from ex vivo biodistribution studies performed at 1 h pi (Table S1) were concordant with the PET/CT imaging data, and both [ 68 Ga]Ga-SB03045 and [ 68 Ga]Ga-SB03058 revealed good FAP-targeting capabilities. Interestingly, contrary to the better in vitro FAPinhibitory performance of nat Ga-SB03058 than nat Ga-FAPI-04, its radioactive equivalent [ 68 Ga]Ga-SB03058 (7.93 ± 1.33 %ID/g) demonstrated a~1.5 fold lower tumor uptake than that of [ 68 Ga]Ga-FAPI-04 (11.9 ± 2.17 %ID/g). The tumor uptake of [ 68 Ga]Ga-SB03045 (11.8 ± 2.35 %ID/g) was comparable to that of [ 68 Ga]Ga-FAPI-04, which was consistent with the results obtained from the FAP-binding assays, where the IC 50 values of [ 68 Ga]Ga-SB03045 and [ 68 Ga]Ga-FAPI-04 were found to be of the same order of magnitude.
Although both tracers displayed fast clearance from most normal organs/tissues, the tumor/background contrast of the thiazolidine derivative [ 68 Ga]Ga-SB03058 was slightly inferior to those of the mono-fluorinated [ 68 Ga]Ga-SB03045 and di-fluorinated [ 68 Ga]Ga-FAPI-04. In addition to a lower tumor uptake, this can be attributed to the higher background uptake of [ 68 Ga]Ga-SB03058, particularly in blood and skeletal muscles (2.07 ± 0.21 and 1.42 ± 0.31 %ID/g, respectively), compared to [ 68 Ga]Ga-SB03045 (1.31 ± 0.43 and 0.83 ± 0.42 %ID/g, respectively) and [ 68 Ga]Ga-FAPI-04 (1.20 ± 0.30 and 0.76 ± 0.20 %ID/g, respectively). The excellent tumor-to-background contrast seen on the PET/CT image of [ 68 Ga]Ga-SB03045 ( Figure 3) and comparable to that previously obtained using [ 68 Ga]Ga-FAPI-04 [30] was further corroborated from the correspondingly comparable tumor/organ uptake ratios obtained in biodistribution studies ( Figure 5 and Table S1). The biodistribution data of mice co-injected with nat Ga-FAPI-04 (0.5 mg/mouse) revealed a~96% reduction in the tumor uptake and further substantiated the in vivo FAP specificity of our lead candidate [ 68 Ga]Ga-SB03045. Interestingly, blocking studies also revealed the bone uptake of [ 68 Ga]Ga-SB03045 to be FAP-specific, as co-injection with excess nat Ga-FAPI-04 resulted in a~97% reduction in bone uptake.
Since [ 68 Ga]Ga-SB03045 exhibited low blood retention at 1 h pi (1.31 ± 0.43 %ID/g), we did not perform imaging and biodistribution studies at later time points to see if tumor uptake can be further improved over time. Instead, derivatives of [ 68 Ga]Ga-SB03045 containing an albumin binder to maximize tumor uptake by virtue of extended blood residence could be exploited in the future, as similar approaches have been reported by others to improve the tumor uptake of [ 68 Ga]Ga-FAPI-04 [38,39].
There are varying and contradictory reports on the association between the uptake intensity of radiolabeled FAPIs and FAP expression [25,[40][41][42][43]. In general, the extent to which the preclinical mouse model is representative of what may be seen in patients and how FAP expression in normal tissues, such as multipotent bone marrow stromal cells, the cervix, and the uterus in humans, would influence the diagnostic performance and radiotherapeutic application of FAP inhibitors still remains ambiguous. More clinical studies may be required in order to ascertain these associations.
The present study design may have certain limitations. The current work is more of a preliminary study in which we were simply trying to compare the potential of two of our novel FAP-targeted pharmacophores for PET/CT imaging against FAPI-04. The data on tracer kinetics is limited, as PET/CT imaging or biodistribution studies were not carried out at multiple time points and no dynamic imaging was performed. Elaborate studies to obtain data on tracer pharmacokinetics and dosimetry to estimate absorbed radiation doses to normal organs/tissues are required in the future if our lead candidate, SB03045, is to be taken for clinical translation. Moreover, FAPI-46 was recently identified as an improvement on FAPI-04 with therapeutic capabilities, due to a longer tumor-retention time than that of FAPI-04 [25]. It would be interesting to incorporate the highly promising FAPI-46 linker into our lead candidate and evaluate the FAP-targeting capabilities of this newly generated agent against its parent compound FAPI-46.

Synthesis of nat Ga-Complexed DOTA-Conjugated FAP-Targeted Ligands
Detailed information on the synthesis, purification, and characterizations of precursors (SB03045 and SB03058) and intermediates and their nat Ga-complexed analogs ( nat Ga-SB03045 and nat Ga-SB03058) is provided in the Supplementary Materials.

Cell Culture
HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The IMPACT Rodent Pathogen Test (IDEXX BioAnalytics, Columbia, MO, USA) verified that the cells were pathogen-free. Detailed information on the generation of HEK293T:hFAP cells has been previously reported by our group [30]. HEK293T:hFAP cells were cultured in DMEM GlutaMAX™ medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 • C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO 2 . Cells were grown until 80-90% confluence and washed with sterile PBS (pH 7.4) and collected.

In Vitro Fluorescence Based Binding Assay
The half-maximal inhibitory concentration (IC 50 ) values of the FAP-targeted ligands were measured using an in vitro enzymatic assay. Briefly, recombinant human FAP (Biolegend, San Diego, CA, USA; 0.2 µg/mL, 50 µL) was added into a Costar clear bottom 96-well plate. PBS and varied concentrations (25 pM to 1 µM) of nat Ga-complexed ligands were added to each well (in duplicate) containing the recombinant human FAP. After being incubated for 30 min at 37 • C, Suc-Gly-Pro-AMC (Bachem, Bubendorf, Switzerland; 20 mM, 50 µL) was added to each well. The velocities of AMC release were measured kinetically at λ ex = 380 nm, λ em = 460 nm at 60 min at 37 • C using a FlexStation 3 Multi-Mode Microplate Reader.

General Procedure for Synthesis of 68 Ga-Complexed Radiotracers
Following our previously published procedures [33], purified [ 68 Ga]GaCl 3 (171 to 260 MBq) in 0.55 mL water was added to a solution of 10 nmol precursor in 0.65 mL HEPES buffer (2M, pH 5.0). The reaction mixture was incubated in a Danby (Guelph, Canada) microwave oven model DMW7700WDB for 1 min at power level 2. After cooling down for 1 min at ambient temperature, the mixture was then purified using HPLC. The eluate fractions containing 68 Ga-labeled radiotracer were collected, diluted with PBS (50 mL), and passed through a C18 Sep-Pak cartridge. 68 Ga-labeled radiotracer trapped on the cartridge was eluted off with ethanol (containing 100 ppm ascorbic acid) and formulated with PBS (containing 100 ppm ascorbic acid) and run on HPLC for quality control before animal studies were performed. The HPLC conditions for preparation of a [ 68 Ga]Ga-SB03045 were a C18 semi-prep column eluted with 10% acetonitrile (containing 0.1% TFA) and 90% deionized water (containing 0.1% TFA) at a flow rate of 4.

Ex Vivo Biodistribution and PET/CT Imaging Studies
Imaging and biodistribution studies were performed using immunodeficient male NRG (NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ) mice. All experiments were conducted according to the guidelines established by the Canadian Council on Animal Care and approved by Animal Ethics Committee of the University of British Columbia. The mice were subcutaneously inoculated with 7.5 × 10 6 HEK293T:hFAP cells in the left dorsal flank. When the tumors grew to 6-8 mm in diameter, the mice were used for PET/CT imaging and biodistribution studies.
PET/CT imaging experiments were carried out using a Siemens Inveon micro PET/CT scanner (Knoxville, TN, USA). Tumor-bearing mice were injected with~4 to 6 MBq (0.02-0.13 nmol) of 68 Ga-labeled tracer through a lateral caudal tail vein under 2.5% isoflurane in oxygen anesthesia, followed by recovery and free roaming in their cage during the uptake period. A 10 min localization CT scan was acquired using 3 overlapping positions to cover each entire mouse. A CT scan was used for attenuation and scatter correction, and anatomical localization. A list mode acquisition was then performed for 15 min at 1 h pi with each mouse under isoflurane sedation. The images were reconstructed using 3D OSEM/MAP iterative methods.
For biodistribution studies, the mice were injected with~1-2 MBq (0.01-0.04 nmol) of the 68 Ga-labeled tracer, using the exact procedures described above. At 1 h post injection, the mice were euthanized by CO 2 inhalation. Blood was withdrawn by cardiac puncture and organs/tissues of interest were collected, weighed, and counted using a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter. The percent injected dose per gram of tissues (%ID/g) for different tracers was calculated.
For blocking studies, HEK293T:hFAP tumor-bearing mice were co-injected with [ 68 Ga]Ga-SB03045 and excess nonradioactive Ga-FAPI-04 (0.5 mg/mouse) as a competitor. Imaging and biodistribution were performed at 1 h pi, similar to the process for the unblocked mice.

Hydrophilicty/LogD 7.4 Measurement
Briefly, 68 Ga-labeled tracers (1.11-1.48 MBq) were aliquoted into vials containing 3 mL of n-octanol and 3 mL of 0.1 M phosphate buffer (pH 7.4) in triplicate. Each vial was then vortexed (1 min) and centrifuged (5000 RPM, 10 min). The n-octanol and aqueous layers were sampled (1 mL) and counted using an automated gamma counter and the LogD 7.4 value was calculated using the following equation:
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28083481/s1. Detailed synthetic procedures and results for the preparation of FAP-targeted ligands and their nat Ga-complexed analogs; Table S1: Biodistribution and tumor/organ uptake ratios of [ 68 Ga]Ga-SB03045, [ 68 Ga]Ga-SB03058 and [ 68 Ga]Ga-FAPI-04 in HEK239T:hFAP tumor-bearing mice; Figure S1: A representative MS spectrum of FAPI-04; Figure S2: A representative MS spectrum of SB03045; Figure S3: A representative MS spectrum of SB03058; Figure S4: A representative MS spectrum of nat Ga-FAPI-04; Figure S5: A representative MS spectrum of nat Ga-SB03045; Figure S6: A representative MS spectrum of nat Ga-SB03058; Figure S7  and Pancreas Centre BC. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Institutional Review Board Statement:
The animal study protocol (A20-0113) was approved by the Animal Ethics Committee of the University of British Columbia on 30 September 2020.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data generated from this study are available in the text and in the Supplementary Materials.